Ethanol resistance saccharomyces cerevisiae gp-01 by protoplast fusion, method for manufacturing thereof, method for manufacturing yeast containing high content of bio organic germanium by using saccharomyces cerevisiae gp-01 and high water soluble sodium metagermanate as a germanium source

ABSTRACT

A biologically pure culture of  Saccharomyces cervisiae  GP-01, and a method for producing  Saccharomyces Cervisiae  GP-01 which is ethanol-resistant and obtained by protoplast fusing  Saccharomyces Cerevisiae  (KCTC 7904) and  Candida Ethanolica  (KCTC 7181). Further, the present invention provides yeast containing the high content of organic bio-germanium (the yeast Ge-32K) and a method for producing the yeast Ge-32K, comprising adding the yeast GP-01 into a solution of sodium metagermanate (Na 2 GeO 3 ) at the volume ratio of 1:0.5˜2; adding 0.1˜0.4 wt % of surfactant, instead of germanium dioxide as used in the prior arts; and cultivating an obtained broth. The obtained yeast Ge-32K contains the higher content of the organic bio-germanium than the conventional yeast as produced by using germanium dioxide.

CROSS REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY

The present application claims all benefits accruing under 35 U.S.C.§365(c) from the PCT International Application PCT/KR2008/007834, withan International Filing Date of Dec. 31, 2008, which claims the benefitof Korean patent application No. 10-2008-0122704 filed in the KoreanIntellectual Property Office on Dec. 4, 2008, the entire contents ofwhich are incorporated herein by reference.

BACKGROUND 1. Technical Field

The present invention relates to yeast which is ethanol resistant andcontains the high content of organic bio-germanium, and the method forproducing the yeast. More specifically, the method comprises producing amutated Saccharomyces cervisiae, i.e., yeast GP-01 by protoplast fusionof Saccharomyces cervisiae (KCTC7904) and candida ethanolica (KCTC7181),the yeast being resistant against the high concentrate ethanol;cultivating the yeast GP-01 with sodium metagermanate (Na₂GeO₃) forgermanium to be inoculated into the yeast GP-01 by thebiotransformation; and finally obtaining a mutated yeast beingethanol-resistant and containing the high content of organicbio-germanium (hereinafter referred to as the yeast Ge-32K).

2. Background Art

Germanium is classified with organic and inorganic germanium (GeO₂). Itis known the inorganic germanium accumulated in the human organs overthe long time leads to the strong toxicity.

Organic germanium is naturally contained in microorganisms, mineralwater and medicinal herbs. Further, it can be bio-synthesized in thefungal body or chemically synthesized. The organic germanium isorganically bound to the proteins in the cell, peptides, etc. and thetherapeutic benefits of the organic germanium for enhancing immunity;suppressing the origination and spread of the tumor cells (metastasis),treating and preventing various chronic diseases, etc. are establishedby numerous papers and scientific journals (Kor. J. Microbiol.Biotechnol., 2007. 35(2):118-127; J. Kor. Soc. Food Sci. Nutr. 2006.35(6):683-689; J. Kor. Soc. Appl. Biol. Chem. 2005.48(3):246-251; ImmuneNetwork. 2006. 6(2):86-92). In addition, the organic germanium iswell-known for anticancer activities by oxygen supply to cells (J.Orthomol. Medicin., 1986. 1:145-148), purification of blood (Medicin.Hyphothesis., 1988. 26:207-215), reinforcement of immunity by activationof NK cell and macrophage (Microbiol. Immunol., 1985. 29:65-74; Kor. J.Microbiol. Biotechnol., 2007. 35(2):118-127); inducement of interferonproduction (Gantokagakutyoho, 9:1976-1980); suppression of generatingand spreading the cancer cell (Cancer and Chemotheraphy. 1985.12(12):2345-2351); suppression of arthritis (Autonomic & AutacoidPharmacol. 2005. 25:129-134); prevention and treatment of variousdiseases such as neuralgia (Biotechnol. Appl. Biochem., 1986.8:379-386), osteoporosis (Germanium; The health and life enhancer.1988); mitigation of high blood pressure (hypertension) andhyperlipidemia cholesterol (Pharmacol., 1990. 41:286-291); alleviationof liver toxin (J. Kor. Soc. Food. Nutr., 1994. 23(4): 581-586); and soforth. Due to the well-known therapeutic benefits, many researches onorganic germanium have been widely undertaken for decades in an attemptto apply it for treatment of various obstinate diseases such as cancerand cardiopathy, etc. in U.S.A., Japan, Europe and Korea (AnticancerRes., 1985. 5(5):479-483).

The organic germanium can be obtained by extracting from medicinal herbslike ginseng, ganoderma, etc., but such a method is difficult to becommercialized due to high cost and low productivity. Further, theorganic germanium can be obtained by a chemical synthesis which includesa reaction of germanium dioxide (GeO₂) with organic acids, but theproduct obtained from the chemical synthesis comprises the inorganicgermanium and thus the use for the foods or medical supplies is limiteddue to the un-established safety (Import Alert by US FDA, 1988).

In fact, the yeast is importantly used in baking, confectionery andbrewery industry for thousands of years. Further, the yeast itself isuseful as a source of nutrition for human being and attractive as a nextgeneration protein source, a single cell protein (SCP) which is ideallycomprised of protein, vitamin, mineral, etc. and characterized in thehigh protein and low fat.

Furthermore, the yeast is the best source of Vitamin B among the singlecells and comprising lots of enzymes etc., which are mainly served invivo metabolism. In this respect, the yeast is used as the healthsupplementary product. Recently, a large number of researches anddevelopments on the health supplement products have been made so thatthe physiological and pharmaceutical benefits by the yeast are obtained.In order to achieve the said object, the development for the yeastcontaining the high contents of organic bio-germanium is highlyrequired.

In the conventional methods, the yeast containing the organicbio-germanium is produced by cultivating the yeast in the mediumincluding inorganic germanium (GeO₂). Due to the drawback that thegrowth of yeast is suppressed in the condition of the medium where thehigh content of the inorganic germanium is contained, it is difficult inthe conventional methods to produce the yeast containing the appropriateamount of organic bio-germanium in aspect of the cost-effective andscale.

To remedy the above drawback, a method separately executing the processof producing the yeast in the form of a pellet and the process ofinoculating germanium dioxide (GeO₂) in the obtained yeast is tried.However, in the above method, the yield of the palletized yeast is toolow to be commercialized due to the ethanol generated during thecultivation of yeast and the content of germanium dioxide (GeO₂)inoculated into the yeast is extremely low.

Also it has been known that the solubility of germanium dioxide (GeO₂)used in the above method is 5.2 g/L at 25° C., which is served as thesuppresser of the growth of yeast (J. Ind. Microbiol. Biotechnol., 1989.4(4):299-306).

-   1. Therefore, it is highly required to invent the method to produce    the ethanol resistant yeast containing the high content of organic    bio-germanium by using the inorganic germanium which has higher    solubility but much lower toxicity than germanium dioxide (GeO₂).

SUMMARY

It is an object of the present invention to provide a yeast GP-01(Saccharomyces cervisiae GP-01, KCTC11399BP) with ethanol resistance inorder to increase the yield of the yeast, and a method for producing theyeast GP-01.

It is another object of the present invention to provide a method forproducing a yeast Ge-32K containing the high content of organicbio-germanium, obtained by bio-synthesis of the mutated saccharomycescervisiae (ethanol resistant yeast GP-01) and sodium metagermanate(Na₂GeO₃).

It is a further object of the present invention to provide the yeastGe-32K containing the high content of organic bio-germanium, obtained bybio-synthesis of the mutated saccharomyces cervisiae (yeast GP-01,KCTC-11399BP) and sodium metagermanate (Na₂GeO₃).

The present invention is related to a mutated saccharomyces cervisiae(yeast GP-01, KCTC11399BP) being ethanol-resistant and having an abilityto produce the organic germanium and a method for producing the yeastGe-32K containing the high content of organic bio-germanium, comprisingcultivating the said yeast GP-01 with sodium metagermanate (Na₂GeO₃).

According to the present invention, a large number of yeast GP-01 can beproduced by protoplast fusion of saccharomyces cerevisiae (KCTC-7904)and candida ethanolica (KCTC 7181). The obtained yeast GP-01 isethanol-resistant, which enables to be cultivated under the high ethanolcondition. The ethanol-resistant yeast GP-01 of the present inventioncan overcome the drawback of the prior arts that the growth yield of theyeast is decreased due to the ethanol generated during the cultivationof yeast.

Furthermore, as the productivity of the yeast GP-01 in cultivation isincreased more than 3 times compared to that of the initial yeast(KCTC-7904), the mass-production of the yeast Ge-32K containing the highcontent of organic bio-germanium can be obtained by using the yeastGP-01 of the present invention.

In the process of inoculating germanium into pelletized yeast GP-01, thepresent invention uses sodium metagerminate (Na₂GeO₃) with solubility of500 g/L, instead of germanium dioxide (GeO₂) commonly used in the priorarts, and thus the rate of bio-transformation of germanium in the yeastis increased owing to the higher solubility of germanium and iontransmissibility, and lower toxicity of germanium than the germaniumdioxide. It is, therefore, possible for the present invention to obtainthe yeast Ge-32K containing the organic bio-germanium that its contentsis 2 or 3 times higher than that of the prior arts where the germaniumdioxide (GeO₂) is used.

Also, in the process of cultivating the yeast GP-01 (KCTC11399BP) withsodium metagerminate (Na₂GeO₃), the present invention adds surfactantfor the organic bio-germanium to be more effectively inoculated into thepelletized yeast by the biotransformation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the bio-germanium content in the yeast GP-01of the present invention according to the variation of pH.

FIG. 2 is a graph showing the bio-germanium content in the yeast GP-01of the present invention according to the change of temperature.

FIG. 3 is a graph showing the bio-germanium content in the yeast GP-01of the present invention according to the variation of concentration ofsurfactant in wt %.

DETAILED DESCRIPTION

According to an embodiment of the present invention relates to a mutatedSaccharomyces cervisiae, i.e., yeast GP-01 (KCTC11399BP) having anability to produce the organic germanium.

The mutated Saccharomyces cervisiae, yeast GP-01 was duly deposited inKorea Collection for Type Culture of Korea Research Institute ofBioscience and Biotechnology (having the address of KRIBB,Gwahak-lo-111, Yuseong-gu-Daejeon 305-808, Republic of Korea) under theAccess number of KCTC 11399BP on October 7, 2008.

More specifically, the embodiment of the present invention provides themutated Saccharomyces cervisiae (yeast GP-01, KCTC 11399BP) withethanol-resistance, wherein the method includes protoplast fusion ofSaccharomyces cervisiae (KCTC7904) with candida ethanolica (KCTC7181)having the effective utilization of ethanol.

Further, an embodiment of the present invention provides the yeastGe-32K containing the high content of organic bio-germanium, which canbe produced by cultivating the yeast GP-01 with sodium metagermanate(Na₂GeO₃) solution so as for the germanium to be inoculated into theyeast by bio-transformation.

The mutated yeast can be obtained by protoplast fusion of Saccharomycescervisiae (KCTC7904) and candida ethanolica (KCTC7181) with a mixedsolution of 1M sorbitol as osmotic pressure stabilizer and 0.5 M of2-mercaptoethanol as reducing agent.

And, the protoplast can be obtained by suspending the pelletized yeastin sorbitol solution, to which a cell wall hydrolase is added, toresolve the cell wall. In this case, the preferable volume ratio of 1 Msorbitol and 2-mercaptoethanol is 1: 2-4, more preferably, 1: 2-3.

Also, the protoplast fusion is accomplished by adding two protoplasts ofSaccharomyces cervisiae (KCTC7904) and candida ethanolica (KCTC7181)into the mixed solution comprised of polyethylene glycol (MW 4000); 10mM of calcium chloride (CaCl₂); and one selected among 1 M sorbitol,sucrose and mannitol.

In this case, the preferable volume ratio of the one selected solution(1 M sorbitol), polyethylene glycol and calcium chloride (CaCl₂) is1:2-4:2-5, more preferably, 1:2-3: 2-4.

An embodiment of the present invention provides a method for producingthe yeast Ge-32K containing the high content of organic bio-germanium.The method is comprised of cultivating the yeast GP-01 with sodiummetagermanate (Na₂GeO₃).

The preferable volume ratio of the yeast GP-01(KCTC 11399BP) and sodiummetagermanate (Na₂GeO₃) is 1:0.5-2, more preferably, 1:1.

In addition, it is preferable to cultivate the yeast GP-01(KCTC 11399BP)with sodium metagermanate (Na₂GeO₃) for 19-21 hours at pH 9˜10 andtemperature 3˜40° C. in order for the germanium to be inoculated intothe yeast by the bio-transformation. The above cultivating step may beaccomplished by the ordinary method of cultivating yeast.

By the said method of the present invention, the yeast Ge-32K containingrelatively high content of organic bio-germanium can be obtained.

Hereinafter, the more detailed methods of the present invention forproducing the yeast GP-01 with ethanol-resistance and for producing theyeast Ge-32K containing the high content of organic bio-germanium areprovided.

1^(st) Process: Producing a protoplast fused yeast withethanol-resistance

A protoplast fused yeast was obtained by fusing and cultivating twoprotoplasts of Saccharomyces cervisiae (KCTC7904) and candida ethanolica(KCTC7181), which were deposited at Korea Collection for Type Culture(KCTC) of Korea Research

Institute of Bioscience and Biotechnology.

In more detail, Saccharomyces cervisiae (KCTC7904) and candidaethanolica (KCTC7181) were cultivated, respectively. The obtainedculture mediums were centrifuged to obtain the pelletized yeasts.

It is preferable for the centrifugations of the culture mediums to becarried out when the optical density (O.D.) of the culture mediums is0.45˜0.50 at early logarithmic phase. The palletized yeasts can beobtained from either each of the mediums of the strains or one mediumwhere the two strains are cultured together.

At the volume ratio of 1:1, the two pelletized yeasts were added in asolution, which 1 M of sorbitol and 0.5 M of 2-mercaptoethanol aremixed, in order to wash the yeasts twice. The washed yeasts were leftduring the pre-determined period and then, a solution containing cellwall hydrolase was added to a broth (containing palletized yeasts, 1M ofsorbitol and 0.5 M of 2-mercaptoethanol) at the volume ratio of 1:1 Theyeasts were suspended in the broth to produce protoplasts.

1 M sorbitol, polyethylene glycol (MW 4000) and 10 mM of calciumchloride (CaCl₂) were added to the broth for the protoplast to be fused.Finally, the broth was centrifuged to obtain the protoplast fusedyeasts.

The protoplast fused yeast was cultivated at an agar culture medium for6˜8 days and staining them to identify the protoplast fused yeast. Afterthe protoplast fused yeast was confirmed by the staining, a singlecolony was collected.

Preferably, the used agar culture medium is composed of 3˜5wt % of yeastextract, 7˜9% of peptone, 7˜9wt % of glucose, 0.1˜0.6 wt % of calciumchloride, 72˜75 wt % of sorbitol and 5˜8 wt % of agar, but is notlimited to the above. Any agar conventionally used in the art can bealso applied.

2^(nd) Process: Producing the ethanol resistant yeast which has a highadaptability against a high concentration of germanium (yeast GP-01,(KCTC 11399BP)), and manufacturing a solution of sodium metagermanate(Na₂GeO₃).

The protoplast fused yeast as obtained from the 1^(st) process wascultivated in the medium containing sodium metagermanate (Na₂GeO₃) toenhance the adaptability against the high concentration of germanium ofthe yeast.

In order to produce the yeast GP-01 (KCTC 11399BP), sodium metagermanate(Na₂GeO₃) was used for the germanium-adaptability. The protoplast fusedyeast as obtained from the 1^(st) process was cultivated in liquidculture medium with 3,000˜20,000 ppm of the germanium for 10˜13 hour.

Preferably, the liquid culture medium is composed of 6˜10 wt % ofethanol, 0.1˜0.5 wt % of peptone, 0.1˜0.4 wt % of yeast extract, 2˜5 wt% of glucose, 0.1˜0.4 wt % of malt extract and 84˜90 wt % of water, butis not limited to the above.

Preferably, the optimum concentration of ethanol is 8˜10 wt % and thecontent of germanium in sodium metagermanate (Na₂GeO₃) is 3,000˜10,000ppm in the liquid culture medium.

In case where the concentration of ethanol is less than 6 wt % or over10 wt %, it may lead to the difficulties with obtaining the optimumquantity of the yeast or collecting the ethanol resistant yeastefficiently. Further, in case where the content of germanium is lessthan 3,000 ppm in sodium metagermanate (Na₂GeO₃), it is difficult tocollect the yeast containing the high content of germanium effectivelyand in case where the content of germanium is over 10,000 ppm in sodiummetagermanate (Na₂GeO₃), it is also difficult to obtain thecost-effective yield.

In the liquid culture medium, the yeasts which are actively-grown andhave the lower content of germanium in the supernatant of the culturewere collected. The collected yeasts were sub-cultured in a plate mediumto isolate and obtain a mutated Saccharomyces cervisiae, i.e., yeastGP-01(KCTC 11399BP).

As the obtained yeast GP-01 has the high resistance against ethanol, itis possible for the yield of the yeast to be increased.

Preferably, the plate medium as used above was composed of 3˜5 wt % ofagar, 10˜12 wt % of yeast extract, 20˜23 wt % of peptone, 20˜23 wt % ofglucose and 39˜42 wt % of ethanol, wherein the ethanol was added beforesolidifying the plate, but is not limited to the above.

The prepared plate was double sealed at the room temperature to preventethanol from being evaporated, after the subculture of the yeasts.

Sodium metagermanate (Na₂GeO₃) used as the inorganic germanium in thepresent invention was synthesized by adding germanium dioxide into oneof sodium carbonate (Na₂CO₃), calcium carbonate (CaCO₃), potassiumcarbonate (K₂CO₃) or potassium bicarbonate (KHCO₃) at the equivalentratio, and sterilizing it. The obtained sodium metagermanate (Na₂GeO₃)is a germanium solution with high solubility and should be prepared,immediately prior to the use in the biological transformation process.

The 2^(nd) Process of the present invention, in order for the yeast tobe mutated, may further include radiation of gamma rays (Co⁶⁰) of2.0˜2.5 KGy (D₁₀ value) for 2˜4 hours into the protoplast fused yeastbefore culturing the yeast in the medium containing sodium metagerminate(Na₂GeO₃).

Where the gamma rays (Co⁶⁰) radiation is less than 2.0 KGy, it may bedifficult to obtain the desired mutated yeast because of too muchcolonies of the mutated strains formed on the plate. And, where gammarays (Co⁶⁰) radiation is more than 2.5 KGy, it may be also difficult toobtain the desired mutated yeast because of too high extinction rate ofthe yeast.

The mutated yeast that is irradiated by gamma rays (Co⁶⁰) as mentionedabove can be used for the subculture after culturing it in the YM brothfor 1 hour and stabilizing the same.

3^(rd) Process: Inflow of inorganic germanium ion into the yeast GP-01

The yeast GP-01 as obtained in the 2^(nd) process was cultivated withthe solution of sodium metagermanate (Na₂GeO₃) for the yeast containingthe high content of organic bio-germanium to be produced.

In order to produce the yeast containing the high content of organicbio-germanium (the yeast Ge-32K), the culture medium prepared in the2^(nd) process was centrifuged to collect the yeast only. And, themedium containing the germanium was prepared by inserting the collectedyeast into the solution of sodium metagermanate (Na₂GeO₃) where thecontent of germanium is 3,000˜10,000 ppm.

The volume ratio of the added yeast and the solution of sodiummetagermanate (Na₂GeO₃) is 1:0.5˜2.0.

The obtained medium containing germanium was cultivated for 19˜21 hoursat pH 9˜10 and 30˜40° C. after adding the 0.01˜0.04 wt % of surfactantThus, the yeast containing high content of organic bio-germanium (theyeast Ge-32K) of an embodiment of the present invention was produced.

The added surfactant can lead to increasing the content of germanium inthe cell by the increased permeability of the cell membrane. Thus, it ispossible for the inorganic germanium to be inoculated into the mutatedyeast, i.e., yeast GP-01(KCTC 11399BP).

Preferably, the surfactant used in an embodiment of the presentinvention is Twin-80, but is not limited to the above.

Where the mixing ratio of the yeast and the solution of sodiummetagermanate (Na₂GeO₃) is less than 1:0.5 or over 1:2, it may lead todecreasing content of bio-germanium in the yeast due to the highviscosity of the suspension including the yeast and the lower diffusionspeed between germanium and yeast cell walls.

Further, where the value of pH is less than 9 or over 10, or thetemperature is below 30° C. or over 40° C., it may lead to decreasingthe contents of bio-germanium in the yeast.

In addition, where the surfactant is added in the culture mediumcontaining germanium at less than 0.1 wt %, the permeability of the cellwall is poor. Also, where the surfactant is added in the culture mediumat over 0.4 wt %, the permeability of membrane is inhibited as thestructure of the yeast cell walls is broken so that the contents ofbio-germanium in the yeast may be decreased to the same contents ofbio-germanium as the case where the surfactant is not added.

After producing the yeast containing the high content of organicbio-germanium, i.e., the yeast Ge-32K, by using the mutatedSaccharomyces cervisiae, i.e., yeast GP-01, the remaining mediumcontaining the germanium can be re-used by filtering with 0.2 μmmembrane filter.

In order to assist your understanding on the present invention,exemplary embodiments of the present invention will be described in moredetail as follows. However, the following examples are proposed forillustrative purposes and those skilled in the art will appreciate thatvarious modifications, additions and/or substitutions are possiblewithout departing from the scope and spirit of the invention which isdefined in the accompanying claims and their equivalents.

PREPARATION EXAMPLE 1 Preparing of Sodium Metagermanate (Na₂GeO₃)Solution

The solution of Sodium Metagermanate (Na₂GeO₃) was prepared bydissolving sodium carbonate (Na₂CO₃, Kanto, Japan) in water, addinggermanium dioxide (GeO₂, PPM GmbH, Germany) at the equivalent ratio of1:1 against the said sodium carbonate, and sterilized the obtainedsolution.

The solubility of the solution of Sodium Metagermanate (Na₂GeO₃)prepared as above is quite high, 500 g/L (25° C.).

EXAMPLE 1 Producing of Protoplast Fused Yeast and of Ethanol ResistantYeast by Using the Protoplast Fused Yeast

Saccharomyces Cerevisiae (KCTC 7904) and Candida Ethanolica (KCTC7181)provided by Korea Collection of Type Culture (KCTC) were cultivated atan Erlenmeyer flask containing YPD culture medium for 10 hours at 30° C.The prepared culture medium was centrifuged at the early logarithmicphase to collect the yeast when the UV spectrophotometer of the yeast is0.5. The collected yeast was washed two times with the sterilizedphysiological saline.

The washed yeast was suspended in a solution where 1M sorbitol and 0.5Mmercapto ethanol are mixed at the volume ratio of 1:2 (the suspensionratio between the yeast and the solution is 1:1) for 30 minutes at 30°C. And then, the solution including the yeast was washed by thecentrifuge.

Then, 200 units of a cell wall degrading enzyme, zymolase (L4025, sigma,USA) was added to the centrifuge-washed solution (containing the yeast,1M sorbitol and mercapto ethanol) at the ratio of 1:1. The obtainedsolution was suspended for 1 hour at 30° C. to produce the protoplast ofthe yeast.

A solution of 1M sorbitol, polyethylene glycol (MW 4000) and 10 mL ofsodium chloride (CaCl₂) at the volume ratio of 1:3:2 was added in theabove solution containing the protoplast fused yeast at the equivalentratio. The mixed solution was fused for 30 minutes at 30° C. to producethe protoplast fused yeast of an embodiment of the present invention.

In order to separate the protoplast fused yeast, the protoplast fusedyeast was cultivated for 1 week in SOS solid culture medium containing4.1 wt % of yeast extract, 8.1 wt % of peptone, 8.1 wt % of glucose, 0.4wt % of sodium chloride, 73.2 wt % of sorbitol and 6.1 wt % of agar.After the 1 week cultivation, the yeast was separated by staining itwith carbol fuchin.

The separated yeast was cultivated for 10 hours in the YM liquid mediumcontaining 10 wt % of ethanol, 0.4 wt % of peptone, 0.2 wt % of yeastextract, 4 wt % of glucose, 0.2 wt % of malt extract and 85.2 wt % ofwater after adding 30 wt % of solution of sodium metagermanate (Na₂GeO₃)containing 10,000 ppm of germanium.

In the liquid culture medium, the yeasts which are actively-grown andhave the lower content of germanium were collected in the supernatant ofthe culture. The collected yeasts were subcultured in a plate mediumcontaining 4.1 wt % of agar (Difco), 11.1 wt % of yeast extract (Genico,Korea), 22.0 wt % of peptone (Difco), 22.0 wt % of glucose (Daesang,Korea) and 40.8 wt % of ethanol to isolate and obtain the mutatedSaccharomyces cervisiae, i.e., yeast GP-01 (KCTC 11399BP).

In order to examine the ethanol resistance of the obtained yeast GP-01,the ethanol, which is the inhibitor of the growth, was added by theconcentrations and the status of the growth of the yeast was checked.

Following Table 1 shows the status of growth of the yeast according toethanol concentrations by measuring the growth three times for eachethanol concentration for accuracy.

TABLE 1 Growth status Concentration of The yeast GP-01 GP-01 of KCTC7904GP-01 (1^(st) (2nd) (3^(rd)) ethanol OD(660 nm, investigation) OD(660OD(660 (%, v/v) 1/20) OD(660 nm, 1/20) nm, 1/20) nm, 1/20) 0 0.10 0.100.13 0.12 6.0 0.07 0.15 0.14 0.20 8.0 0.06 0.14 0.20 0.23 10.0 0.05 0.160.19 0.24 15.0 — 0.12 0.08 0.15

Ethanol is generated while the microorganism is cultivated, but inhibitstheir growth.

As shown in the above Table 1, the growth rate of the mutatedSaccharomyces cervisiae, yeast GP-01(KCTC 11399BP) was increased in thepresence of ethanol upon comparing the case in the absence of ethanol.In case where the concentration of ethanol was 6-10%, the best growthrate of the yeast GP-01 was obtained.

In this respect, according to an embodiment of the present invention,the ethanol-resistant yeast, i.e., yeast GP-01 (KCTC 11399BP) which hasthe increased growth rate even in the presence of ethanol can beobtained.

Furthermore, upon comparing the yield of the yeast GP-01 of anembodiment of the present invention with the yield of initial yeast(Saccharomyces Cerevisiae, KCTC 7904), the yield of the yeastGP-01(KCTC-11399BP) of an embodiment of the present invention was morethan 3 times increased.

EXAMPLE 2 Change of Contents of Germanium in Yeast GP-01 byConcentrations of the Solution of Sodium Metagermanate (Na₂GeO₃)

The broth medium containing the mutated Saccharomyces Cerevisiae, yeastGP-01 (KCTC11399BP), was centrifuged to collect the palletized yeast.The collected pelletized yeast was added in each of the solutions ofsodium metagermanate (Na₂GeO₃) with different concentrations ofgermanium. The prepared solutions were cultivated for 20 hours at pH 10and temperature 40° C.

In order to determine the content of bio-germanium in the yeast GP-01, 1g of the cultivated and dried yeast GP-01 was added in 30 ml of nitricacid. The obtained sample was heated to be decomposed. After adjustingpH value to 6, 1 ml of the sample was mixed with 1 ml of phenylfluoroneand 1 ml of cyclo nucleic acid. The obtained sample was stationary for30 hours at 30° C. and then determined the content of germanium in theyeast GP-01 by using UV spectrophotometer at 525 nm.

Also, as a control, the same procedure described in Example 2 wasperformed, except that a solution of germanium dioxide (GeO₂) was usedinstead of sodium metagermanate (Na₂GeO₃).

Following Table 2 shows the comparison of the content of bio-germaniuminoculated into the yeast GP-01 and the initial Saccharomyces Cerevisiae(KCTC 7904) according to the content of germanium in the solutions ofsodium metagermanate (Na₂GeO₃) vs the solution of germanium dioxide(GeO₂).

TABLE 2 Bio-germanium content Contents of inoculated into the yeastGP-01 (ppm) Germanium GeO₂ solution Na₂GeO₃ solution in the Yield (%)Yield (%) solution (ppm) KCTC7904 GP-01 KCTC7904 GP-01 KCTC7904 GP-01KCTC7904 GP-01 1,000 780 870 78 87 — — — — 2,000 870 1,786 44 89 — — — —3,000 980 2,670 33 89 950 4,640 32 155 4,000 1,430 3,850 36 96 1,5006,840 38 171 5,000 1,500 4,753 30 95 2,370 9,700 47 194 10,000 — — 4,50013,000 45 130 15,000 — — 5,730 16,500 38 110

Upon comparing the amounts of the inoculated bio-germanium by thesolutions, Table 2 shows that in case where the amounts of germanium inthe solutions are the same, the more amounts of the bio-germanium wereinoculated into the yeasts in the solution of sodiummetagermanate(Na₂GeO₃) than that in the solution of germanium dioxide(GeO₂) solution.

Also, upon comparing the amount of the inoculated bio-germanium by theyeasts, Table 2 shows that the more amount of the bio-germanium wasinoculated into the mutated Saccharomyces Cerevisiae (yeast GP-01, KCTC11399BP) of the present invention, than the initial SaccharomycesCerevisiae (KCTC 7904).

Where the content of germanium in sodium metagermanate (Na₂GeO₃) is3,000˜10,000 ppm, the most amount of inorganic germanium ion wasinoculated into the yeasts. In addition, where the content of germaniumin the solution is 5,000 ppm, the yield of the germanium to beinoculated reached the highest.

EXAMPLE 3 Comparison of the Content of the Inoculated Bio-Germanium inthe Yeast GP-01 According to the Mixing Ratio Between the PalletizedYeast and the Solution of Inorganic Germaniums (GeO₂ and Na₂GeO₃)

The broth containing the ethanol-resistant yeast GP-01 as prepared inthe Example 1 was centrifuged to collect the yeast. The collected yeastwas added in the solutions which contain sodium metagermanate (Na₂GeO₃)and germanium dioxide but have different mixing ratios of the saidsodium metagermanate (Na₂GeO₃) and germanium dioxide. The obtainedsolutions were cultivated for 20 hours at pH 10 and temperature 40° C.

Also, as a control, the same procedure described in Example 3 wasperformed, except that a solution of (inorganic) germanium dioxide(GeO₂) was used instead of sodium metagermanate (Na₂GeO₃).

The contents of germanium in the yeast GP-01 (KCTC 11399BP) was measuredby the same method as Example 2.

Table 3 shows the comparison of the bio-germanium contents in the yeastGP-01 (KCTC 11399BP) by the mixing ratios of the yeast and germaniumdioxide (GeO₂), and the yeast and sodium metagermanate (Na₂GeO₃).

TABLE 3 Bio-germanium content in yeast Mixing ratio GP-01 (ppm) (yeastGP-01:Ge solution) GeO₂ Solution Na₂GeO₃ Solution   1:0.2 1,920 4,350  1:0.5 5,670 14,800 1:1 4,780 12,600 1:2 2,500 9,760 1:3 1,700 8,720

Table 3 shows that the most amounts of bio-germanium were inoculatedinto the yeast where the mixing ratio between the yeast and theinorganic germanium is 1:0.5˜2. Further, the content of bio-germaniumwas increased more than 2-4 times when adding sodium metagermanate(Na₂GeO₃) solution than adding inorganic germanium (germanium dioxide).

EXAMPLE 4 Optimal Conditions for Inoculating Inorganic Germanium Ioninto the Yeast

The broth containing the ethanol-resistant yeast GP-01 as prepared inthe Example 1 was centrifuged to collect the yeast. At the ratio of1:0.5, the collected yeast was added in the solution of sodiummetagermanate (Na₂GeO₃) having 5,000 ppm of germanium. After addingsurfactant (Twin-80), the obtained solution was cultivated for 20 hoursby various conditions for pH, temperature or the broth containinggermanium.

The pH was regulated by using 1N NaOH, and a number of tests wererepeatedly performed under different temperatures and concentrations ofsurfactant.

When measuring the content of germanium, the broth without adding thesurfactant was used as a control.

The measurement of content of bio-germanium in the yeast GP-01(KCTC11399BP) of the present invention was made by the same method asExample 2.

Table 4 shows the amounts of germanium ion inoculated into the yeast bythe variation of pH, temperature and concentration of surfactant(Twin-80).

TABLE 4 Bio-germanium Bio-germanium Concentration of Bio-germaniuminoculated temperature inoculated Surfactant inoculated pH (ppm) ( ° C.)(ppm) (Twin-80, wt %) (ppm) 4.0 2,190 20 3,102 Control 1,720 5.0 3,25430 9,648 0.1 8,743 6.0 5,320 35 13,250 0.2 16,250 6.5 5,790 40 14,7000.3 12,530 7.0 9,750 45 8,350 0.4 9,670 8.0 10,700 50 4,564 0.5 7,6249.0 15,200 — — 0.6 5,670 10.0 16,300 — — 0.7 4,830 11.0 9,320 — — 0.82,700

Table 4 shows the optimum condition to inoculate the inorganic germaniuminto the yeast is comprised of pH 8˜10, temperature at 30˜40° C., and0.1˜0.4 wt % of surfactant in the broth containing germanium, morepreferably, pH 10, temperature at 40° C. and 0.2 wt of surfactant.

Variation of the content of inoculated bio-germanium by pH, temperatureand the concentration of inorganic germanium can be shown in FIGS. 1, 2and 3.

1. A biologically pure culture of Saccharomyces cervisiae GP-01.
 2. Amethod for producing Saccharomyces cervisiae GP-01, comprisingprotoplast-fusing Saccharomyces Cerevisiae (KCTC 7904) and CandidaEthanolica (KCTC 7181).
 3. The method according to claim 2, wherein theprotoplast-fusing comprises obtaining protoplasts of the SaccharomycesCerevisiae (KCTC 7904) and the Candida Ethanolica (KCTC 7181) by addinga solution comprised of 1M of sorbitol and 0.5M of 2-mercaptoethanol tothe Saccharomyces Cerevisiae KCTC 7904 and the Candida Ethanolica (KCTC7181).
 4. The method according to claim 3, wherein the protoplast fusionfurther comprises fusing the protoplasts by adding the protoplasts intoa solution comprised of with 1M of sorbitol, polyethylene glycol (MW4000) and 10 mM of calcium chloride.
 5. A method for preparing a yeastGe-32K containing organic bio-germanium, comprising adding Saccharomycescervisiae GP-01 to a solution containing sodium metagermanate-(Na₂GeO₃)and culturing the Saccharomyces cervisiae GP-01 in the solutioncontaining the sodium metagermanate (Na₂GeO₃).
 6. The method accordingto claim 5, wherein the sodium metagermanate (Na₂GeO₃) is prepared by(i) dissolving a selected one among sodium carbonate (Na₂CO₃), calciumcarbonate (CaCO₃) and potassium bicarbonate (KHCO₃) in water and addinggermanium dioxide (GeO₂) to the solution prepared by said (i) theequivalent volume ratio.
 7. The method according to claim 5, wherein amixing volume ratio between the Saccharomyces cervisiae GP-01 and thesolution containing the sodium metagermanate (Na₂GeO₃) is 1:0.5-2.0. 8.The method according to claim 6, wherein the culturing is performed for19˜21 hours at pH 9˜10 and temperature 30˜40° C.
 9. A yeast containingorganic bio-germanium produced by the method of claim
 5. 10.Saccharomyces cervisiae GP-01 obtained by the method of claim
 2. 11(New). The method according to claim 3, wherein the protoplast fusionfurther comprises fusing the protoplasts by adding the protoplasts intoa solution comprised of polyethylene glycol (MW 4000), calcium chlorideand one selected from the group consisting of sorbitol, sucrose andmannitol.